Figure 2.

sLIGHT and sFasL show additive killing effect but LIGHT in contrary to FasL functions independently of mitochondrial cytochrome c release. (a) Mouse motoneurons were cultured for 24 h and treated (or not) with sLIGHT (100 ng/ml), recombinant human soluble FasL (sFasL, 100 ng/ml in the presence of 1 μg/ml enhancer antibody), or a combination of both. Motoneuron survival was assessed 48 h after treatment. Note that the enhancer antibody used to cross-link sFasL had no effect on motoneuron survival when added alone or with sLIGHT (not shown)(n=4, ^***P<0.001). (b) Quantification of phospho-p38 kinase fluorescence in Hb9∷GFP motoneurons treated (or not) with sLIGHT in the presence or the absence of SB203580 (10 μM) for 1 h. Treatment with sFasL served as a control.¹⁵ Confocal fluorescence imaging and ImageJ image analysis were used to determine the nuclear mean fluorescence intensity of p38 in Hb9∷GFP neurons (a.u, arbitrary unit, ^*P<0.05, ^**P<0.01). (c) Motoneurons were maintained in culture for 24 h and treated or not with sLIGHT (100 ng/ml) and SB203580 (10 μM). Survival was determined 48 h later and expressed relative to survival in the absence of any treatment. (d and e) Hb9∷GFP motoneurons were incubated or not with 100 ng/ml of sLIGHT or sFasL, fixed and immunostained with anti-cytochrome c antibody 30 h later. sLIGHT-treated Hb9∷GFP motoneuron shows the same punctuate labeling of mitochondria as non-treated motoneurons (not shown), whereas following Fas activation a proportion of motoneurons show diffuse labeling of cytochrome c. Scale bar, 10 μm. (e) The percentage of Hb9∷GFP motoneurons showing diffuse versus punctuated cytochrome c labeling was determined by direct counting under fluorescence microscope (n=4). (f) Survival assay was done as in (c). The Bax inhibitory peptide V5 was used at the concentration of 50 μM. (g) Mouse motoneurons were co-electroporated with an equimolar ratio of a vector coding for EGFP and a vector coding for either Bcl-Xl, a dominant negative form of Bid (tⁿ-Bid) or an empty vector (e.v) and cultured for 24 h before being treated or not with sLIGHT or sFasL. The survival of EGFP-positive motoneurons for each combination of vectors was expressed as a percentage of surviving motoneurons in the absence of sLIGHT or sFasL. Histograms show mean values±S.D. of at least three independent experiments, each done in triplicate